HO-1 suppressor as a diagnostic and prognostic test for dementing diseases

ABSTRACT

The invention relates to an improved method for predicting the onset of, diagnosing, prognosticating and/or treating dementing diseases. The method comprises determining the level of heme oxygenase-1 suppressor (HOS) activity and/or factor in tissue or body fluid obtained from a patient, and comparing said level with the corresponding level of HOS activity and/or factor in corresponding tissue or body fluid obtained from at least one control person. The tissue or body fluid is suitably blood, plasma, lymphocytes, cerebrospinal fluid, urine, saliva, epithelia or fibroblasts. The method is useful where the dementing disease is any of Alzheimer Disease, Age-Associated Cognitive Decline, Mild Cognitive Impairment, Parkinson disease with dementia, Progressive Supranuclear Palsy, Vascular (i.e. multi-infarct) Dementia, Lewy Body Dementia, Huntington&#39;s Disease, Down&#39;s syndrome, normal pressure hydrocephalus, corticobasal ganglionic degeneration, multisystem atrophy, head trauma, neurosyphilis, Creutzfeld-Jacob disease and other prion diseases, HIV and other encephalitides, and metabolic disorders such as hypothyroidism and vitamin B12 deficiency. The method may also prove useful in differentiating the “pseudodementia” of depression from Alzheimer disease.

This application claims the benefit of International Application No.PCT/CA1/01066, filed Jul. 25, 2001 and published on Jan. 31, 2002 asInternational Publication No WO 02/08449 A2 (incorporated herein byreference in its entirety), which claims the benefit of United StatesProvisional Application No. 60/220,813 filed on Jul. 25, 2000.

FIELD OF THE INVENTION

Applicant's related U.S. Pat. No. 6,210,895 (Apr. 3, 2001), hereinincorporated by reference, relates to a method for predicting,diagnosing, and/or prognosticating dementing diseases such as AlzheimerDisease (AD) and Age-Associated Cognitive Decline (AACD). The inventionrelates to improved methods for predicting, diagnosing, prognosticatingand/or treating dementing diseases such as Alzheimer Disease (AD) andAge-Associated Cognitive Decline (AACD) or Mild Cognitive Impairment(MCI) as well as methods and reagents to facilitate the study of thecause and progression of these diseases.

BACKGROUND OF THE INVENTION

Alzheimer Disease (AD) is a neurodegenerative disease which causesdementia. The terms “Alzheimer Disease” and “Alzheimer's Disease” areboth utilized in the art, these terms being equivalent and are usedinterchangeably here and elsewhere. The period from first detection ofAD to termination can range from a few years to 15 years, during whichtime the patient progressively suffers loss of both mental function andcontrol of bodily functions. There is significant variability in theprogress of the disease. While the majority of patients have a gradual,inexorable progression (losing on average 3 to 4 points on the 30 pointFolstein mini-mental state score annually), approximately 30% of ADcases have a prolonged stable initial plateau phase lasting severalyears, as described in Haxby et al. (1992), herein incorporated byreference. A subgroup of patients has a fulminant, rapidly progressivedownhill course over several years, as described in Mann et al. (1992),herein incorporated by reference. Other patients (about 10% of cohorts)remain slowly progressive, showing only gradual decline from year toyear, as described in Grossi et al. (1988), herein incorporated byreference. The pathological, chemical and molecular bases of thisheterogeneity remain undetermined. Recognition of the variability of ADprogression represents an important clinical insight, and may explainthe diagnostic difficulties presented by “atypical” cases.

Attempts at predicting the onset of AD or monitoring its progressionhave met with limited success. While in certain cases, there is afamilial manifestation of the disease, it appears that the majority ofAD cases are non-familial, and until recently (see below), no simplegenetic marker for the disease had been determined. Much research hasfocused on the protein beta-amyloid, deposits of which are found in thebrains of AD victims.

However, recently, as described in our related U.S. patent (U.S. Pat.No. 6,210,895Apr. 3, 2001 and publication (Schipper et al., 2000), bothherein incorporated by reference, we have devised a diagnostic method,useful in the prediction, diagnosis, and prognostication of AD,AACD/MCI, and related neurological diseases. This diagnostic method isbased on the determination that patients suffering from AD have asignificantly lower concentration of heme oxygenase-1 (HO-1) in theirlymphocytes and plasma, and, accordingly, a significantly lowerconcentration of ribonucleotide sequences encoding HO-1 in theirlymphocytes.

HO-1: Heme oxygenase-1 (HO-1) is an enzyme that catalyses the rapiddegradation of heme to biliverdin in brain and other tissues. This 32kDa member of the heat shock protein superfamily contains a heat-shockelement in its promoter region and is rapidly up regulated in responseto oxidative stress, metal ions, amino acid analogues, sulfhydrylagents, and hyperthermia. In response to oxidative stress, induction ofHO-1 may result in protection of cells by catabolizing pro-oxidantmetalloporphyrins, such as heme, into bile pigments (biliverdin,bilirubin), with free radical scavenging capabilities. Heme and otherintracellular ferrous iron chelates are capable of converting hydrogenperoxide to the highly cytotoxic hydroxyl radical.

Using immunostaining techniques in conjunction with laser scanningconfocal microscopy, intense HO-1 immunoreactivity in neurons andastrocytes of post-mortem hippocampus and temporal cortex derived fromAD subjects has been observed, whereas neural HO-1 staining was faint ornon-existent in the hippocampus and temporal cortex of control specimensmatched for age and post-mortem interval, as noted in Schipper et al.(1995), herein incorporated by reference. Furthermore, consistentco-localization of HO-1 to neurofibrillary tangles and senile plaques inthe AD specimens has been demonstrated. Finally, robust 32 kDa bandscorresponding to HO-1 were observed by Western blotting of proteinextracts derived from AD temporal cortex and hippocampus after SDS-PAGE,whereas control HO-1 bands were faint or absent. These results indicatethat HO-1 is significantly over-expressed in neurons and astrocytes ofAD hippocampus and cerebral cortex relative to control brains andsupport the contention that AD-affected tissues are experiencing chronicoxidative stress.

AACD/MCI: AACD and MCI are terms used to identify individuals whoexperience a cognitive decline that falls short of dementia. These termsare equivalent, MCI being a more recently adopted term, and are usedinterchangeably throughout this application. Satisfaction of criteria(World Health Organization) for this diagnosis requires a report by theindividual or family of a decline in cognitive function, which isgradual, and present at least 6 months. There may be difficulties acrossany cognitive domains (although memory is impaired in the vast majorityof cases), and these must be supported by abnormal performance onquantitative cognitive assessments for which age and education norms areavailable for relatively healthy individuals (i.e., the patient iscompared to normal subjects his/her own age). Performance must be atleast 1 SD below the mean value for the appropriate population on suchtests. Neither dementia, nor significant depression or drug effects maybe present. No cerebral or systemic disease or condition known to causecerebral cognitive dysfunction may be present. In Applicant'sexperience, all patients who were classified as CDR.5 (“questionabledementia”) on the Clinical Dementia rating scale and who met theseexclusions, also met the criteria for AACD/MCI. About ⅓ of Alzheimer'spatients have had a clearly definable period of isolated memory deficitwhich preceded their more global cognitive decline, as noted by Haxby etal. (1992), herein incorporated by reference. Using AACD/MCI criteriawhich look at other domains in addition to memory, the percentage withan identifiable prodrome is likely higher. Fortunately, not all AACD/MCIindividuals seem to decline. It appears that a significant number ofthese subjects show a stable, non-progressive memory deficit on testing.

Related Disorders: Determining HO-1 concentration can also assist inpredicting, diagnosing, or prognosticating other dementing diseaseshaving similar manifestations and/or in distinguishing such diseasesfrom AD. Such other diseases include Parkinson disease with dementia,Progressive Supranuclear Palsy, Vascular (i.e. multi-infarct) Dementia,Lewy Body Dementia, Huntington's Disease, Down's syndrome, normalpressure hydrocephalus, corticobasal ganglionic degeneration,multisystem atrophy, head trauma, neurosyphilis, Creutzfeld-Jacobdisease and other prion diseases, HIV and other encephalitides, andmetabolic disorders such as hypothyroidism and vitamin B12 deficiency.The method may also prove useful in differentiating the “pseudodementia”of depression from Alzheimer disease.

The determination of a relationship between HO-1 levels and ADrepresents a very significant advance in this field, and may be utilizedfor the development of methods of predicting, diagnosing in its veryearly stage, and prognosticating AD and other dementing diseases.However, identification of the factor(s) and mechanism(s) which controlHO-1 expression in the normal versus the diseased state are needed, toprovide even earlier diagnosis, as well as therapeutic methods andreagents or substances, and methods and reagents for the study of AD andother dementing diseases. In addition, the reduction or absence of HO-1in patients suffering from AD represents a negative test, and,particularly for the purposes of diagnosis, it would be more desirableto have a positive indicator of disease, i.e. a factor whose presence(rather than absence) correlates with disease. Further, the decrease inHO-1 expression may represent an effect, rather than a cause of AD andother dementing diseases, therefore the identification of factor(s) andmechanism(s) which control HO-1 expression in the normal versus thediseased state are also needed to identify components and events whichhave an active causative role in the onset and progression of thesediseases.

SUMMARY OF THE INVENTION

It is a goal of the present invention to provide improved methods forpredicting, diagnosing, prognosticating and/or treating AD and otherdementing diseases, as well as methods and reagents to facilitate thestudy of the cause and progression of these diseases.

Advantageously, embodiments of this invention provide an easilyadministered blood or cerebrospinal fluid test which is used to predict,diagnose, or prognosticate AD and other dementing diseases.

One aspect of the present invention is a heme oxygenase-1 suppressor(HOS) factor, wherein said factor attenuates the increase in the levelof heme oxygenase-1 (HO-1). In an embodiment, such an increase occurs inresponse to exposure to an experimental agent or treatment which iscapable of increasing the level of HO-1. For example, such experimentalagents or treatments comprise exposure to any one or more of oxidativestress, metal ions, amino acid analogues, sulfhydryl agents (e.g.,cysteamine, homocysteine), interleukin-1β, tumour necrosis factor-α(TNF-α) and hyperthermia.

Another aspect of the present invention is a method for assessingdementing diseases in a patient which comprises: determining the levelof heme oxygenase-1 suppressor (HOS) factor or activity, in tissue or abody fluid obtained from a patient and comparing said level of HOSfactor or activity with the corresponding level of HOS factor oractivity in corresponding tissue or body fluid obtained from at leastone control person, whereby if said level of HOS factor or activity isgreater than said corresponding level of HOS factor or activity in saidtissue or body fluid obtained from at least one control person then saidpatient suffers from a dementing disease wherein such method is used topredict the onset of, diagnose, or prognosticate dementing diseases.

Yet another aspect of the present invention is a diagnostic method fordifferentiating, in a patient suffering from a dementing disease,between a dementing disease which is HO-1-dependent and a dementingdisease which is HO-1-independent, said method comprising: determiningthe level of heme oxygenase-1 suppressor (HOS) factor or activity, intissue or a body fluid obtained from a patient suffering from adementing disease and comparing said level of HOS factor or activitywith the corresponding level of HOS factor or activity in correspondingtissue or body fluid obtained from at least one control person, wherebyif said level of HOS factor or activity differs significantly from saidcorresponding level of HOS factor or activity in said tissue or bodyfluid obtained from at least one control person then said patientsuffers from a dementing disease which is HO-1-dependent, and if saidlevel of HOS factor or activity does not differ significantly from saidcorresponding level of HOS factor or activity in said tissue or bodyfluid obtained from at least one control person then said patientsuffers from a dementing disease which is HO-1-independent.

In an embodiment, another aspect of the present invention is a methodfor differentiating the pseudodementia of depression from otherdementing diseases in a patient which comprises: determining the levelof heme oxygenase-1 suppressor (HOS) factor or activity, in tissue orbody fluid obtained from a patient and comparing said level of HOSfactor or activity with the corresponding level of HOS factor oractivity in corresponding tissue or body fluid obtained from at leastone control person whereby if said level of HOS factor or activity isgreater than said corresponding level of HOS factor or activity in saidcorresponding tissue or body fluid obtained from at least one controlperson then said patient suffers from a dementing disease other than thepseudodementia of depression wherein such method is used todifferentiate the pseudodementia of depression from other dementingdiseases.

The dementing diseases assessed using the methods described aboveinclude, but are not limited to, Alzheimer Disease, Age-AssociatedCognitive Decline, Mild Cognitive Impairment, Parkinson disease withdementia, Progressive Supranuclear Palsy, Vascular (i.e. multi-infarct)Dementia, Lewy Body Dementia, Huntington's Disease, Down's syndrome,normal pressure hydrocephalus, corticobasal ganglionic degeneration,multisystem atrophy, head trauma, neurosyphilis, Creutzfeld-Jacobdisease and other prion diseases, HIV and other encephalitides, andmetabolic disorders such as hypothyroidism and vitamin B12 deficiency.Further, as noted above, the methods may also prove useful indifferentiating the “pseudodementia” of depression from Alzheimerdisease.

Examples of the above mentioned tissue or body fluids include, but arenot limited to, blood, plasma, lymphocytes, cerebrospinal fluid, urine,saliva, epithelia, and fibroblasts.

The above-mentioned control tissue or body fluid, for example, may beobtained from at least one normal age-matched control person or from thepatient at another time, in an embodiment, at an earlier time.

Yet another aspect of the present invention is a method for assaying thelevel of heme oxygenase-1 (HO-1) suppressor (HOS) factor or activity ina sample which comprises: exposing the sample to a cell culturesubjecting the cell culture to exposure to an experimental agent ortreatment which may increase the level of HO-1 protein or mRNA encodingHO-1; determining the level of HO-1 protein or mRNA encoding HO-1; andcomparing said level of HO-1 protein or mRNA encoding HO-1 with acorresponding control level of HO-1 protein or mRNA encoding HO-1;

whereby the level of said HO-1 protein or mRNA encoding HO-1 inverselycorrelates with the level of HOS factor or activity.

The present invention also provides evidence for the existence of aputative heme oxygenase-1 (HO-1) suppressor (HOS) factor in the samplesderived from a patient suffering a dementing disease, as well as apartially purified fraction comprising HOS activity and a correspondingputative HOS factor.

Accordingly, a further aspect of the present invention is a method forassaying the level of heme oxygenase-1 (HO-1) suppressor (HOS) factor oractivity in a sample which comprises: exposing the sample to a cellculture subjecting the cell culture to exposure to an experimental agentor treatment which may increase the level of HO-1 protein or mRNAencoding HO-1; determining the level of HO-1 protein or mRNA encodingHO-1; and comparing said level of HO-1 protein or mRNA encoding HO-1with a corresponding control level of HO-1 protein or mRNA encodingHO-1; whereby the level of said HO-1 protein or mRNA encoding HO-1inversely correlates with the level of HOS factor or activity.

The above-mentioned corresponding control level of HO-1 protein or mRNAmay be obtained, for example, by assaying the level of HO-1 protein ormRNA in a corresponding cell culture which has been subjected toexposure to the above-mentioned experimental agent or treatment, but hasnot been exposed to the above-mentioned sample prior to exposure to theabove-mentioned experimental agent or treatment.

Additional aspects of the present invention are polyclonal andmonoclonal antibodies which recognize the HOS factor, as well ashybridoma cells which produce the latter monoclonal antibodies.

Yet a further aspect of the present invention is a method for assayingthe level of heme oxygenase-1 (HO-1) suppressor (HOS) factor or activityin a sample comprising: exposing said sample to an antibody whichrecognizes the HOS factor; isolating immune complexes; and determiningthe level of HOS factor or activity in the immune complex.

Since HOS affects the levels of HO-1 mRNA and protein, therefore theinvention also contemplates a method for assaying the level of HOSactivity or factor using a reporter construct comprising transcriptionalregulatory element(s) (e.g., a promoter region) of the HO-1 geneoperably linked to a suitable reporter gene.

Accordingly, a further aspect of the present invention is a method forassaying the level of heme oxygenase-1 (HO-1) suppressor (HOS) activityin a sample comprising: exposing said sample to a reporter construct,wherein said reporter construct comprises the HO-1 promoter region and areporter gene, wherein said reporter gene encodes a protein whichpossesses a detectable reporter activity; determining the level of saidreporter activity, and comparing said level of said reporter activitywith a corresponding control level of said reporter activity; wherebythe level of said reporter activity inversely correlates with the levelof HOS factor or activity.

The above-mentioned control level of reporter activity may be obtained,for example, by measuring the reporter activity produced by acorresponding reporter construct that has not been exposed to theabove-mentioned sample.

The HOS activity of the present invention may also be used for theelucidation of other factors and mechanisms involved in the onset andprogression of AD and other dementing diseases. These factors andmechanisms may yield therapeutic agents and methods, as well ascontribute to our understanding of the molecular events which areinvolved in the onset and progression of AD and other dementingdiseases.

Therefore, a further aspect of the present invention is a method forscreening a candidate compound for the presence of an inhibitor oractivator of HOS activity or HOS factor comprising: exposing saidcandidate compound to a sample known to comprise HOS activity or HOSfactor; assaying the level of HOS activity or HOS factor using a methodselected from the group consisting of: (a) a method for assaying thelevel of heme oxygenase-1 (HO-1) suppressor (HOS) factor or activity ina sample which comprises: exposing the sample to a cell culture;subjecting the cell culture to exposure to an experimental agent ortreatment which may increase the level of mRNA encoding HO-1;determining the level of HO-1 protein or mRNA encoding HO-1; andcomparing said level of HO-1 protein or mRNA encoding HO-1 with acorresponding control level of HO-1 protein or mRNA encoding HO-1;whereby the level of said HO-1 protein or mRNA encoding HO-1 inverselycorrelates with the level of HOS factor or activity; (b) a method forassaying the level of heme oxygenase-1 (HO-1) suppressor (HOS) factor oractivity in a sample comprising: exposing said sample to an antibodywhich recognizes the HOS factor; isolating immune complexes anddetermining the level of HOS factor or activity in the immune complex;and (c) a method for assaying the level of heme oxygenase-1 (HO-1)suppressor (HOS) factor or activity in a sample comprising: exposingsaid sample to a reporter construct, wherein said reporter constructcomprises the HO-1 promoter region and a reporter gene, wherein saidreporter gene encodes a protein which possesses a detectable reporteractivity and determining the level of said reporter activity; andcomparing said level of said reporter activity; with a correspondingcontrol level of said reporter activity; whereby the level of saidreporter activity inversely correlates with the level of HOS factor oractivity and comparing said level of HOS activity or HOS factor with acorresponding control level of HOS activity or HOS factor in acorresponding control sample, wherein said control sample comprises saidsample known to comprise HOS activity that has not been exposed to saidcandidate compound.

A further aspect of the present invention is a commercial packagecomprising means for determining the level of heme oxygenase-1 (HO-1)suppressor (HOS) factor or activity, in tissue or body fluid obtainedfrom a patient, and instructions for comparing said level of HOS factoror activity with an established standard of the corresponding HOSactivity in corresponding control tissue or body fluid. Such controltissue or body fluid, for example, may be obtained from at least onenormal age-matched control person or from the patient at another time,in an embodiment, at an earlier time.

Since levels of HO-1 mRNA, protein and/or activity as well as HOS factorand/or activity may be altered in patients suffering from a dementingdisease, inhibitors or activators of HOS factor or HOS activityrepresent potential substances or compounds which may be utilized forthe treatment of a dementing disease.

Accordingly, a further aspect of the present invention is a compound forthe treatment of a dementing disease, wherein the compound alleviatesthe dementing disease by increasing or decreasing the level of hemeoxygenase-1 (HO-1) mRNA, protein or activity.

A further aspect of the present invention is a compound for thetreatment of a dementing disease, wherein the compound alleviates thedementing disease by increasing or decreasing the level of hemeoxygenase-1 (HO-1) suppressor (HOS) factor or activity.

Yet a further aspect of the present invention is a pharmaceuticalcomposition for the treatment of a dementing disease which comprises thesubstance or compound described above in admixture with a suitablepharmaceutically acceptable diluent or carrier.

Yet a further aspect of the present invention is a method of treating adementing disease in a patient, comprising administering to said patientthe compound or pharmaceutical composition described above in an amounteffective to treat a dementing disease, wherein said method results inthe alleviation of the dementing disease by increasing or decreasing thelevel of heme oxygenase-1 (HO-1) mRNA, protein or activity.

Yet a further aspect of the present invention is a method of treating adementing disease in a patient, comprising administering to said patientthe compound or pharmaceutical composition described above in an amounteffective to treat a dementing disease, wherein said method results inthe alleviation of the dementing disease by increasing or decreasing thelevel of heme oxygenase-1 (HO-1) suppressor (HOS) factor or activity.

Yet a further aspect of the present invention is a use of theabove-mentioned compound or pharmaceutical composition for the treatmentof a dementing disease.

Yet a further aspect of the present invention is a commercial packagecontaining as an active pharmaceutical ingredient the compound orpharmaceutical composition described above together with instructionsfor its use in the treatment of a dementing disease.

The substance or compound, composition, method and commercial packagenoted above may, for example, be utilized for the treatment of adementing disease selected from the group consisting of AlzheimerDisease, Age-Associated Cognitive Decline, Mild Cognitive Impairment,Parkinson disease with dementia, Progressive Supranuclear Palsy,Vascular (i.e. multi-infarct) Dementia, Lewy Body Dementia, Huntington'sDisease, Down's syndrome, normal pressure hydrocephalus, corticobasalganglionic degeneration, multisystem atrophy, head trauma,neurosyphilis, Creutzfeld-Jacob disease and other prion diseases, HIVand other encephalitides, and metabolic disorders such as hypothyroidismand vitamin B12 deficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A–1C Northern analysis of HO-1 mRNA implicating the presence of acirculating HO-1 suppressor (HOS) factor in sporadic AD, as described inExample 1. Control GAPDH bands used to ensure uniformity of RNA loadingare depicted below the HO-1 bands.

FIG. 2 depicts tabular results of studies of demographics and HOSactivity in normal young control (NYC), normal elderly control (NEC),mild cognitive impairment (MCI) and sporadic Alzheimer disease (AD)subjects, as described in Example 2. Suppression by 24 h incubation inhuman plasma of CSH-induced (880 μM×6 h) glial HO-1 mRNA band (Northernblot) relative to CSH-treated astrocytes grown in standard culturemedia; 0=0–25% suppression, 1=26–50% suppression, 2=51–75% suppression,3=76–100% suppression. HOS=HOS activity MMSE=Folstein Mini-mental StateExam Score; Cortisol=Plasma cortisol levels (nMol/L); ADMeds=cholinesterase inhibitors used for treatment of Alzheimer disease;E400 and E800=400 and 800 units vitamin E, respectively; C500=500 mgvitamin C.

FIG. 3 depicts graphical results of HOS activity in normal control (NC),mild cognitive impairment (MCI) and sporadic Alzheimer disease (AD)subjects. HOS activity=percentage suppression (quartiles) by 24 hincubation in human plasma of CSH-induced (880 μM×6 h) glial HO-1 mRNAband (Northern blot) relative to CSH-treated astrocytes grown instandard culture media, as described in Example 3.

FIG. 4 depicts analysis of plasma cortisol levels (mean±SD) in normalcontrol (NC), mild cognitive impairment (MCI) and sporadic Alzheimerdisease (AD) subjects (panel A), as described in Example 4. ( )=numberof cases per group. Differences between groups are not statisticallysignificant (1-way ANOVA). Correlation between plasma cortisol levelsand HOS activity in the MCI (panel B) and AD (panel C) groups is notsignificant (linear regression analysis).

FIG. 5 is a Northern analysis of HO-1 mRNA demonstrating the effects ofsample storage time and antioxidant exposure on plasma HOS activity, asdescribed in Example 8. C=Control (untreated) astrocyte cultures,CSH=cysteamine-treated astrocyte culture, AD=Alzheimer, MCI=MildCognitive Impairment, NEC=normal elderly control, N=normal subject onantioxidants. Control GAPDH bands are used as noted in FIG. 1.

FIGS. 6A–6B Northern analysis of HO-1 mRNA demonstrating the effects ofplasma dilution on HOS activity, as described in Example 6.

FIG. 7 is a Northern analysis of HO-1 mRNA demonstrating the effect ofheat treatment on HOS activity, as described in Example 7. Control GAPDHbands are used as noted in FIG. 1.

FIG. 8 is a Northern analysis of HO-1 mRNA demonstrating the partialpurification of HOS factor by heparin-agarose chromatography, asdescribed in Example 8. Control GAPDH bands are used as noted in FIG. 1.

FIG. 9 is a Northern analysis of HO-1 mRNA demonstrating further HOSpurification of the heparin agarose eluate by Concanavalin-A (Con-A)Agarose affinity column chromatography, as described in Example 9.Control GAPDH bands are used as noted in FIG. 1.

FIG. 10 is a Northern analysis of HO-1 mRNA demonstrating further HOSpurification of the heparin agarose-conconavalin A eluate derived from 4pooled AD plasma samples (29 cc starting material) on a Superose™ 12 HRFPLC Column, as described in Example 10. Control GAPDH bands are used asnoted in FIG. 1.

FIG. 11 presents graphical results of relative protein concentrations inSuperose™ 12 HR FPLC Column fractions derived from pooled AD plasmasamples described in FIG. 10, as described in Example 10. Arrow denotesprotein concentration in fraction (number 20–22) exhibiting robust HOSactivity.

FIG. 12 depicts results of a chromatogram from a function test ofSuperose™ 12 HR FPLC 1-cm diameter column (Catl. # 17-0538-01, Lot #8283034) [Amersham Pharmacia Biotech, Inc Quebec Canada] using standardprotein mixtures, as described in Example 10.

FIG. 13 is a Northern analysis of HO-1 mRNA demonstrating the effects ofNEC and AD plasma on astrocyte HO-1 mRNA induction by multiple stimuli.The HOS bioassay was performed as described for FIG. 1. Northern blotsfor HO-1 mRNA (top) and respective GAPDH mRNA (bottom) are shown.Control GAPDH bands are used as noted in FIG. 1.

DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION

Applicant has devised an improved diagnostic method, useful in theprediction, diagnosis, and prognostication of AD, AACD/MCI, and relatedneurological diseases, as well as methods and reagents which are usefulin the treatment and study of AD, AACD/MCI, and related neurologicaldiseases. These methods are based on the discovery that patientssuffering from AD have an activity and corresponding factor in theirplasma which significantly suppresses the expression of heme oxygenase-1(HO-1). This HO-1 suppressor activity is assayed via the inability toupregulate the concentration of nucleotide sequences encoding HO-1, inresponse to exposure to a suitable experimental agent or treatment, in asuitable cell culture system pre-incubated with a tissue or body fluidderived from patients suffering from AD or other dementing diseases.This suppressor activity and corresponding factor shall be referred toas HO-1 suppressor (HOS) activity and factor, respectively.

Applicant has identified an activity, namely HOS activity, which ispresent in tissue or body fluids derived from patients suffering from ADas well as possibly those suffering from other dementing diseases, butis absent in normal age-matched control subjects. This activity may bedetected in a tissue or body fluid obtained from these patients.Examples of possible sources of suitable tissue or body fluids includeblood, plasma, lymphocytes, cerebrospinal fluid, urine, saliva,epithelia (such as skin epithelia) and fibroblast cell lines derivedfrom patients.

An aspect of the invention is a HOS activity, which is an activity thatsuppresses the upregulation of HO-1 expression. Such upregulationoccurs, for example, following exposure to an experimental agent ortreatment which is, in the absence of HOS activity, capable ofincreasing HO-1 expression, as detected by increases in HO-1 protein orHO-1 mRNA. In patients suffering from AD, as well as possibly thosesuffering from other dementing diseases, HOS activity suppresses theexpression of HO-1, which is expressed at significantly higher levels inlymphocytes and possibly other non-neural tissue or body fluids innormal aged-matched control subjects.

A further aspect of the invention is a method of assaying HOS activityin a sample. Examples of possible sources of suitable samples includetissues and body fluids such as, for example, blood, plasma,lymphocytes, cerebrospinal fluid, urine, saliva, epithelia andfibroblast cell lines derived from patients, or fractions derived fromthese samples. The assay involves exposing the sample to be tested to acell culture that is capable of undergoing an induction in HO-1expression in response to exposure to a certain experimental agent ortreatment. An example of such a cell culture is a rat astroglialculture, however, many other useful possibilities exist. Examples ofsuch exposure to an experimental agent or treatment include exposure tooxidative stress, metal ions, amino acid analogues, sulfhydryl agents,interleukin-1β, tumour necrosis factor-α (TNF-α) and hyperthermia.Examples of suitable sulfhydryl reagents include, but are not limitedto, cysteamine and homocysteine. Following such exposure to anexperimental agent or treatment, the level of HO-1 protein or HO-1 mRNAmay be detected using suitable methods. The level of HO-1 may forexample be detected by an immunoassay. The level of HO-1 mRNA may forexample be detected by Northern analysis using an appropriate probe(s).Detection of HO-1 mRNA of greater sensitivity may be achieved forexample using the reverse transcriptase-polymerase chain reaction(RT-PCR) method, described in Abraham (1998) and Mawal et al. (2000),both herein incorporated by reference. The activity assay may be adaptedto a large scale level for analyzing a large number of samplessimultaneously, possibly in a suitable array format, possibly with theautomated execution (e.g., by robotics) of some or all of the stepstherein.

A further possibility may be the development of a reporter-based assayfor assaying HOS activity. Such an assay may involve the preparation ofa suitable reporter construct, e.g. comprising a transcriptionalregulatory element, such as the 5′ untranslated promoter region, of theHO-1 gene, operably linked to a suitable reporter gene, i.e., capable ofregulating the expression of a suitable reporter gene. Such a constructmay additionally comprise the 3′ untranslated region of the HO-1 gene oranother suitable 3′ sequence. In another embodiment, the construct maycomprise an in frame fusion of a suitable reporter gene within the openreading frame of the HO-1 gene. The reporter gene may be chosen as suchto facilitate the detection of its expression, e.g. by the detection ofthe presence and/or activity of its gene product. Many such suitablereporters may be used, which provide detectable signals. Most preferredembodiments in this class are those that provide a convenientlydetectable signal, which may be detected by, for example, spectroscopicmethods. Examples of suitable reporter genes include those encodingluciferase, beta-galactosidase, green fluorescent protein, alkalinephosphatase, chloramphenicol acetyltransferase, as well as others. Sucha reporter construct may be introduced into a suitable system capable ofexhibiting an increase in the level of expression of the reporter genein response to exposure to an experimental agent or treatment which iscapable of increasing HO-1 expression as noted above. Such an assaywould also be adaptable to a possible large scale, high-throughput,automated format as noted above, and would allow more convenientdetection due to the presence of its reporter component.

Using methods of assaying HOS activity as described above, applicant hasdetermined that the level of HOS activity in a sample decreases with theincreasing dilution of the sample, suggesting that HOS activity isattributed to the presence of a corresponding HOS factor. Using the sameassay methods, applicant has further determined that pre-heating thesample to be tested abrogates HOS activity, suggesting that HOS activityis attributed to a protein or complex of proteins. Since, to applicant'sknowledge, glucocorticoids are the only known suppressors of HO-1expression (Lavrovsky et al., 1996; Deramaudt et al., 1999), thediscovery of a protein-like HOS factor is novel. Applicant has furtherdemonstrated that cortisol levels are not increased in AD or MCI sampleswith respect to normal samples, thus demonstrating that suppression ofHO-1 expression in AD and MCI samples is not attributed toglucocorticoids, but rather, is a result of the activity of a(non-glucocorticoid) HOS factor.

Applicant has accomplished a partial purification of HOS activity andtherefore HOS factor using one or multiple chromatographic methods insequential fashion. An example of a suitable chromatographic method isaffinity chromatography using a heparin-agarose matrix or aconcanavalin-A (Con-A) agarose matrix or gel filtration chromatographyusing for example a Superose™-12 matrix. Applicant has accomplishedfurther purification of HOS factor using heparin-agarose, concanavalin-A(Con-A) agarose and Superose™-12 chromatography, in sequence, furthersuggesting that HOS factor comprises a protein or complex of proteins,and, based on binding to the Con-A matrix, likely comprises aglycoprotein, in an embodiment, a mannoprotein. This suggests that HOSactivity and the corresponding HOS factor may be obtained in a morehighly purified form using various chromatographic methods. Suchpurification is for example shown in FIG. 11, where the peak of HOSactivity elutes later that most of the protein in the sample, thusindicating that the Superose™-12 column has removed the majority ofprotein contaminants from the HOS factor-containing sample. Calibrationof the column using known protein molecular weight standards (FIG. 12)indicates that HOS factor is a protein or complex of proteins having anapproximate molecular weight in the range of 80–100 kDa, in anembodiment, having a molecular weight of approximately 90 kDa. Thesedata thus provide further support that HOS factor is a protein-likemolecule. Applicant has further shown that HOS factor and associated HOSactivity are stable during prolonged storage.

Accordingly, the invention further provides a HOS factor, as describedabove.

Applicant has further demonstrated that HOS activity is not due tosimple antioxidant behavior, since both AD and normal plasma exhibitequivalent levels of partial suppression of the HO-1 mRNA response to apro-oxidant, for example, menadione. Further, typical doses ofantioxidants have no effect on the induction of HO-1 mRNA expression,and exposure of multiple, high dose, antioxidants only results inpartial suppression.

A further aspect of the present invention is an improved diagnosticmethod, potentially useful in the prediction, diagnosis, andprognostication of AD, AACD/MCI, and related neurological diseases. Thisdiagnostic method is based on the detection of HOS activity, using forexample the assay methods described above, in a tissue or body fluidobtained from a patient. Because the presence of HOS activity precedesany decrease in HO-1 expression in a patient, this diagnostic methodprovides an even earlier diagnosis of AD, AACD/MCI, and relatedneurological diseases. In addition, the immunodetection of HOS factor oractivity (see below) may provide an improved method of diagnosis overthe detection of decreases in HO-1 expression using methods such asNorthern analysis or the reverse transcriptase-polymerase chain reaction(RT-PCR) method, described in Abraham (1998) and Mawal et al. (2000),both herein incorporated by reference. Further, the correlation of thepresence of HOS activity with the disease state represents a positivetest for diagnosis. This is more desirable than a negative test, usedfor diagnosis based on the reduction or absence of HO-1 expression in apatient suffering from one of the dementing diseases described above.

It is known in the art that certain dementing diseases, for example, AD,correlate with changes in HO-1 levels while others do not. Suchdementing diseases may be categorized as HO-1-dependent andHO-1-independent. As described in the instant application, such changesin HO-1 levels are a result of changes in the levels of HOS factor oractivity. Therefore, the invention further relates to methods, reagents,compounds and commercial packages to differentiate a dementing diseasewhich exhibits a significantly altered level of HO-1 protein, HO-1 mRNA,HOS factor, or HOS activity, i.e., an HO-1-dependent dementing disease,from a dementing disease which does not exhibit such a significantlyaltered level of HO-1 protein, HO-1 mRNA, HOS factor, or HOS activity,i.e., an HO-1-independent dementing disease. The term “significantly” asused here means that the levels are altered from control levels beyondthe range of experimental error, as known in the art.

The HOS activity of the present invention may also be used to developtherapeutic agents and methods for the treatment of AD and otherdementing diseases. Since the appearance of HOS activity correlates withthe presence of the disease state, the HOS activity and HOS factor isexpected to play a causative role in the onset and/or progression of ADand other dementing diseases. Therefore, identification of factors ormechanisms which inhibit or activate HOS activity may be utilized forthe development of therapeutic agents and methods for the treatment ofAD and other dementing diseases. If an increase in HOS activity is acausative event in the onset and/or progression of AD and otherdementing diseases, an inhibitor of HOS activity is expected to havetherapeutic potential. Conversely, an activator of HOS activity isexpected to represent an upstream causative agent of the onset and/orprogression of AD and other dementing diseases, which may provide evenearlier and improved methods of diagnosis. Further, all factors whicheffect HOS activity will lead to a better understanding of themechanisms of the onset and/or progression of AD and other dementingdiseases, and ultimately contribute to the development of improvedtherapeutic methods and agents. In addition, other factors which affectlevels of HO-1 mRNA, protein and activity are also useful to theinvention, similar to the above, and are thus a further aspect of theinvention.

Accordingly, it is a further aspect of the present invention to providea HOS activity-based screening method to identify putative compoundswhich either inhibit or augment HOS activity. Such screening may beperformed using for example the HOS activity assays described above, andmay be adapted to a large scale, and possibly automated format. Such amethod may comprise exposing a known HOS activity-containing sample tothe compound to be tested, and subsequently determining the level of HOSactivity present, which is then compared to a control sample that wasnot exposed to the compound to be tested. In a high-throughput,automated format, this screening method may be used for the rapidanalysis of libraries containing a large number of compounds for theireffects on HOS activity. In an embodiment, examples of such librariesinclude chemical libraries prepared by combinatorial synthesis.

The partially purified fraction comprising HOS factor and HOS activity,obtained, for example, from heparin-agarose and/or Con-A agarose and/orSuperose™-12 column chromatography, may be used to immunize a smallmammal, e.g., a mouse or a rabbit, in order to raise antibodies whichrecognize this activity. In an embodiment the above mentioned fractionis obtained from sequential heparin-agarose, Con-A agarose andSuperose™-12 column chromatography. Accordingly, a further aspect of theinvention provides an antibody that recognizes the HOS factor of theinvention.

An antibody of the invention is either polyclonal or monoclonal.Antibodies may be recombinant, e.g., chimeric (e.g., constituted by avariable region of murine origin associated with a human constantregion), humanized (a human immunoglobulin constant backbone togetherwith hypervariable region of animal, e.g., murine, origin), and/orsingle chain. Both polyclonal and monoclonal antibodies may also be inthe form of immunoglobulin fragments, e.g., F(ab)′₂, Fab or Fab′fragments. The antibodies of the invention are of any isotype, e.g., IgGor IgA, and polyclonal antibodies are of a single isotype or a mixtureof isotypes.

Antibodies against the HOS factor of the present invention are generatedby immunization of a mammal with a partially purified fractioncomprising HOS factor. In an embodiment the above mentioned fraction isobtained from sequential heparin-agarose, Con-A agarose and Superose™-12column chromatography. Such antibodies may be polyclonal or monoclonal.Methods to produce polyclonal or monoclonal antibodies are well known inthe art. For a review, see Harlow and Lane (1988) and Yelton et al.(1981), both of which are herein incorporated by reference. Formonoclonal antibodies, see Kohler and Milstein (1975), hereinincorporated by reference.

The antibodies of the invention, which are raised to a partiallypurified fraction comprising HOS factor of the invention, are producedand identified using standard immunological assays, e.g., Western blotanalysis, dot blot assay, or ELISA (see, e.g., Coligan et al. (1994),herein incorporated by reference). The antibodies are used in diagnosticmethods to detect the presence of a HOS factor and activity in a sample,such as a tissue or body fluid. The antibodies are also used in affinitychromatography for obtaining a purified fraction comprising the HOSfactor and activity of the invention.

Accordingly, a further aspect of the invention provides (i) a reagentfor detecting the presence of HOS factor and activity in a tissue orbody fluid; and (ii) a diagnostic method for detecting the presence ofHOS factor and activity in a tissue or body fluid, by contacting thetissue or body fluid with an antibody of the invention, such that animmune complex is formed, and by detecting such complex to indicate thepresence of HOS factor and activity in the sample or the organism fromwhich the sample is derived.

Those skilled in the art will readily understand that the immune complexis formed between a component of the sample and the antibody, and thatany unbound material is removed prior to detecting the complex. It isunderstood that an antibody of the invention is used for screening asample, such as, for example, blood, plasma, lymphocytes, cerebrospinalfluid, urine, saliva, epithelia and fibroblasts, for the presence of HOSactivity.

For diagnostic applications, the reagent (i.e., the antibody of theinvention) is either in a free state or immobilized on a solid support,such as a tube, a bead, or any other conventional support used in thefield. Immobilization is achieved using direct or indirect means. Directmeans include passive adsorption (non-covalent binding) or covalentbinding between the support and the reagent. By “indirect means” ismeant that an anti-reagent compound that interacts with a reagent isfirst attached to the solid support. Indirect means may also employ aligand-receptor system, for example, where a molecule such as a vitaminis grafted onto the reagent and the corresponding receptor immobilizedon the solid phase. This is illustrated by the biotin-streptavidinsystem. Alternatively, a peptide tail is added chemically or by geneticengineering to the reagent and the grafted or fused product immobilizedby passive adsorption or covalent linkage of the peptide tail.

Such diagnostic agents may be included in a kit which also comprisesinstructions for use. The reagent is labeled with a detection meanswhich allows for the detection of the reagent when it is bound to itstarget. The detection means may be a fluorescent agent such asfluorescein isocyanate or fluorescein isothiocyanate, or an enzyme suchas horse radish peroxidase or luciferase or alkaline phosphatase, or aradioactive element such as ¹²⁵I or ⁵¹Cr.

Accordingly, a further aspect of the invention provides a process forpurifying, from a tissue or body fluid, the HOS factor of the invention,which involves carrying out antibody-based affinity chromatography withthe tissue or body fluid, wherein the antibody is an antibody of theinvention.

For use in a purification process of the invention, the antibody iseither polyclonal or monoclonal, and preferably is of the IgG type.Purified IgGs are prepared from an antiserum using standard methods(see, e.g., Coligan et al. (1994), herein incorporated by reference).Conventional chromatography supports, as well as standard methods forgrafting antibodies, are described in, e.g., Harlow and Lane (1988),herein incorporated by reference, and outlined below.

Briefly, a tissue or body fluid, such as plasma from a patient sufferingfrom AD, preferably in a buffer solution, is applied to a chromatographymaterial, preferably equilibrated with the buffer used to dilute thetissue or body fluid so that the HOS factor of the invention (i.e., theantigen) is allowed to adsorb onto the material. The chromatographymaterial, such as a gel or a resin coupled to an antibody of theinvention, is in either a batch form or a column. The unbound componentsare washed off and the antigen is then eluted with an appropriateelution buffer, such as a glycine buffer or a buffer containing achaotropic agent, e.g., guanidine HCl, or high salt concentration (e.g.,3 M MgCl₂). Eluted fractions are recovered and the presence of theantigen is detected, e.g., by measuring the absorbance at 280 nm.

A further aspect of the present invention is a diagnostic imagingmethod, which comprises introducing into a biological system, anantibody of the invention, which is used in conjunction with anappropriate detection system to identify areas where HOS factor oractivity is present or absent.

The following examples are provided in order to illustrate the methodsof the present invention and are not meant to limit the scope of theinvention.

EXAMPLE 1 Determination of the Presence of HOS Activity in PlasmaDerived from AD Patients

Whole blood is collected from normal elderly (N1, N2) subjects orpatients with probable sporadic AD (AD1, AD2) in heparinized tubes. Thisis then layered over a Ficoll Paque™ density gradient and centrifuged at1800 rpm for 20 minutes. The top plasma layer is then collected andsaved for incubation with rat astroglia as described below. Thelymphocyte fractions are collected and used for the isolation of mRNAfor Northern analysis as described below.

Determination of lymphocyte HO-1 mRNA levels: Lymphocyte fractions wereobtained by differential centrifugation of whole blood on Ficoll Paque™gradients as described above. Cytoplasmic RNA was isolated from thelymphocytes using an acid guanidinium thiocyanate-phenol-chloroformextraction method, as described by Chomczynski et al. (1997),Biotechniques 22(3):550–3, herein incorporated by reference. Sixmicrograms of RNA was denatured and size-separated by electrophoresis on1% agarose/formaldehyde gels. RNA integrity was confirmed by ethidiumbromide staining. The RNA was transferred onto Hybond-N™ filter paperand covalently cross-linked to the membrane by UV light for two minutes.The hybridization probe (HO-1; 1.0 kb) was prepared by random primingusing the Random Primer DNA Labeling System, as described by Feinberg etal. (1984), herein incorporated by reference. Prehybridization wasperformed for 12 hours at 42° C. in a buffer containing formamidedeionized, 5× Denhardt's reagent, 6×SSPE and 0.5% SDS. The hybridizationbuffer consisted of the prehybridization buffer without 5× Denhardt'sreagent, and ³²P-labelled denatured DNA probe, as described in Noonberget al. (1994), herein incorporated by reference. Equal loading of RNAwas confirmed by hybridization with a cDNA for the (housekeeping) gene,glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All washes wereperformed under stringent conditions (1×SSC and 0.2% SDS for 45 minutesat room temperature, 0.4×SSC and 0.2% SDS for 15 minutes at 65 C). TheRNA hybridizing with cDNA probes was visualized by autoradiography usingan intensifying screen at −80 C, as described in Church et al. (1984),herein incorporated by reference.

As noted in our related U.S. patent (U.S. Pat. No. 6,210,895; Apr. 13,2001) and publication (Schipper et al., 2000), both herein incorporatedby reference, and as reiterated in Panel A of FIG. 1, lymphocytesisolated from normal subjects N1 and N2 exhibit significant levels ofHO-1 mRNA (lanes 1 and 2), which is not detectable in lymphocytesisolated from AD patients AD1 and AD2 (lanes 3 and 4).

Assay of Plasma HOS Activity Via the Induction of HO-1 Expression UponCysteamine (CSH) Treatment of Rat Astroglia

Brain cell cultures: Rat astroglia were prepared as described inSchipper et al. (1999), herein incorporated by reference, as follows:

Pregnant Sprague-Dawley rats were obtained from Charles River BreedingFarms. Primary neural cell cultures were prepared from 1-day oldneonates by mechanoenzymatic dissociation of cerebral tissue or bodyfluid as previously described by Chopra et al., (1997), hereinincorporated by reference. Cells were grown in Ham's F-12 andhigh-glucose DMEM (50:50 vol/vol) supplemented with 10 mM HEPES. 5%heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum,and penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively). Thecells were plated in 75-cm² tissue or body fluid culture flasks at adensity of 1×10⁶ cells/ml. Cultures were incubated at 37 C in humidified95% air/5% CO₂ for 6 h, at which time they were vigorously shaken 20–30times with replacement of fresh medium to remove adherentoligodendroglia and microglia from the astrocytic monolayers. Thecultures were then incubated under the above-mentioned conditions for 6days, at which time >98% of the cells composing the monolayer wereastroglia, as determined by immunohistochemical labeling for theastrocyte-specific marker glial fibrillary acidic protein, as describedby Chopra et al. (1995), herein incorporated by reference. Theseastroglia cultures were grown under different conditions and subjectedto different treatments (see below), and subsequently mRNA was isolatedfor Northern analysis of HO-1 mRNA levels as described in Schipper etal. (1999), herein incorporated by reference, as follows:

RNA isolation and Northern analysis: Cultured astrocytes were harvestedusing a rubber policeman, and cytoplasmic RNA was isolated using an acidguanidinium thiocyanate/phenol/chloroform extraction method, asdescribed by Chomczynski and Sacchi (1987), herein incorporated byreference. Ten micrograms of RNA was denatured and size-separated byelectrophoresis on 1% agarose/formaldehyde gels. RNA integrity wasconfirmed by ethidium bromide staining. The RNA was transferred ontoHybond-N™ filter paper and covalently cross-linked to the membrane by UVlight for 2 min. The hybridization probe (HO-1; 1.0 kb) was prepared byrandom primer-generated double-strand DNA probes using the random primerDNA labeling system, as described by Feinberg and Vogelstein (1984),herein incorporated by reference. Prehybridization was performed for 12h at 42 C in a buffer containing formamide-deionized, 5× Denhardt'sreagent, 6× saline-sodium phosphate-EDTA, and 0.5% sodium dodecylsulfate (SDS). The hybridization buffer consisted of theprehybridization buffer without 5× Denhardt's reagent and ³²P-labeleddenatured DNA probe, as described by Noonberg et al. (1994), hereinincorporated by reference. Equal loading of RNA was confirmed byhybridization with a cDNA for the (housekeeping) gene,glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or 18S mRNA. Allwashes were performed under stringent conditions [1×saline-sodiumcitrate (SSC) and 0.2% SDS for 45 min at room temperature, 0.4×SSC and0.2% SDS for 15 min at 65 C, and 0.1×SSC and 0.2% SDS for 15 min at 65°C.]. The RNA hybridizing with cDNA probes was SDS for 15 min at 65 C,and 0.1×SSC and 0.2% SDS for 15 min at 65 C]. The RNA hybridizing withcDNA probes was visualized by autoradiography using an intensifyingscreen at −80 C, as described by Church and Gilbert (1984), hereinincorporated by reference. Resulting bands on the autoradiograph wereanalyzed using a phosphorimager S1 densitometer. Densitometry data werenormalized by calculating the ratios of the HO-1 mRNA signals to controlGAPDH or 18S mRNA signals.

FIG. 1, panel A: Northern blot of lymphocyte HO-1 mRNA (and controlGAPDH mRNA) derived from 2 normal elderly individuals (N1, N2) and 2patients with probable sporadic AD AD1, AD2). As noted in our relatedU.S. Patent (U.S. Pat. No. 6,210,895; Apr. 13, 2001 and publication(Schipper et al., 2000), both herein incorporated by reference,lymphocyte HO-1 mRNA bands are visible in the controls (lanes 1 and 2),and not detectable (lanes 3 and 4) in the AD subjects, indicating thepresence of HOS activity in the latter.

Using the methods described above, Northern analysis of HO-1 mRNA levelsof rat astroglia grown under different conditions and subjected todifferent treatments was performed, the results of which are shown inpanels B and C of FIG. 1. Panel B: Control (unchallenged) rat astrogliagrown in standard culture media for 6 days exhibit faint or no HO-1 mRNAbands (lanes 5–7). Cysteamine (CSH) treatment (880 μM×6 hr) inducesrobust HO-1 mRNA bands in these cells (lanes 8–10). Twenty-four hourincubation of the rat astroglia with the plasma derived from the same 2normal subjects (N1, N2; lanes 11–12) and the 2 AD patients (AD1,AD2lanes 13–14) noted above (see panel A) has no appreciable affect onbaseline HO-1 mRNA levels. Panel C: In contrast to plasma derived fromthe 2 normal subjects (lanes 15–24), undiluted plasma from the 2 ADpatients markedly suppresses the rat astroglial HO-1 mRNA response toCSH (lanes 25–27; 30–32). Dilution of the AD plasma (1:9 in standardculture media; “10%”) greatly diminishes its inhibitory effect onCSH-induced HO-1 mRNA expression (lanes 28–29; 33–34). Therefore, thereexists in the plasma of AD patients an HOS activity, which is notpresent in the plasma of normal subjects, and which is assayable by thedetermination of HO-1 mRNA levels in rat astroglia incubated with therelevant plasma sample and subjected to CSH treatment.

EXAMPLE 2 Demographics and HOS Activity in Normal Young Control (NYC),Normal Elderly Control (NEC), Mild Cognitive Impairment (MCI) andSporadic Alzheimer Disease (AD) Subjects

Results are shown in tabular form in FIG. 2. Suppression by 24 hincubation in human plasma of CSH-induced (880 μM×6 h) glial HO-1 mRNAband (Northern blot) relative to CSH-treated astrocytes grown instandard culture media; 0=0–25% suppression, 1=26–50% suppression,2=51–75% suppression, 3=76–100% suppression. HOS=HOS activity;MMSE=Folstein Mini-mental State Exam Score Cortisol=Plasma cortisollevels (nMol/L). AD Meds=cholinesterase inhibitors used for treatment ofAlzheimer disease. E400 and E800=400 and 800 units vitamin E,respectively C500=500 mg vitamin C. HOS activity was assayed asdescribed in Example 1. Measurement of cortisol levels were performedusing the GammaCoat [I-125] Cortisol Radioimmunoassay (RIA) Kit based onthe competitive binding principles of RIA.

EXAMPLE 3 HOS Activity in Normal Control (NC), Mild Cognitive Impairment(MCI) and Sporadic Alzheimer Disease (AD) Subjects

Results are shown in FIG. 3. HOS activity=percentage suppression(quartiles) by 24 h incubation in human plasma of CSH-induced (880 μM×6h) glial HO-1 mRNA band (Northern blot) relative to CSH-treatedastrocytes grown in standard culture media. HOS activity was assayed asdescribed in Example 1.

EXAMPLE 4 Plasma Cortisol Levels (Mean±SD) in Normal Control (NC), MildCognitive Impairment (MCI) and Sporadic Alzheimer Disease (AD) Subjects

Results are shown in FIG. 4. Panel A shows mean (±SD) plasma cortisollevels of NC, MCI and AD subjects. ( )=number of cases per group.Differences between groups are not statistically significant (1-wayANOVA). Correlations between plasma cortisol levels and HOS activity inthe MCI (panel B) and AD (panel C) groups are not significant (linearregression analysis). Although glucocorticoids are known suppressors ofthe HO-1 gene, these data indicate that elevated cortisol levels are notresponsible for HOS activity in the MCI and AD plasma.

EXAMPLE 5 Effects of Sample Storage Time and Antioxidant Exposure onPlasma HOS Activity

Results are shown in FIG. 5. HOS activity was assayed as described inExample 1. C=Control (untreated) astrocyte cultures,CSH=cysteamine-treated astrocyte culture, AD=Alzheimer, MCI=MildCognitive Impairment, NEC=normal elderly control, N=normal subject onantioxidants. Protease inhibitors (Complete Protease Inhibitor Cocktail,Cat. # 1836153, Roche, Mannheim) were added to all plasma samples priorto freezing. HOS activity is retained in AD and MCI plasma samplesstored at −85 C for up to 15 months. In normal subjects, low-dosevitamin E (400 U/day), a dose of vitamin E commonly taken by ADpatients, does not affect the astrocyte HO-1 mRNA response to CSH (N1).In normal individuals, exposure to multiple, very high-dose antioxidantspartially attenuates the glial HO-1 mRNA response to CSH (N2, N3).

EXAMPLE 6 Effects of Plasma Dilution on HOS Activity

Plasma HOS activity was assayed via the determination of HO-1 mRNAlevels in treated rat astroglia as described in Example 1. In this case,the effects of plasma dilution were examined, as documented in FIG. 6.

Lane 1: Absence of HO-1 mRNA in unchallenged rat astrocytes grown instandard culture media. Lane 2: Cysteamine (CSH 880 μM×6 h)inducesstrong HO-1 mRNA bands in cultured astroglia grown in standard media.Lane 3: Absence of HO-1 mRNA bands in unchallenged astrocytes grown inAlzheimer (AD) plasma (A: patient 1; B: patient 2). Lanes 4–6: undilutedAD plasma markedly suppresses the HO-1 mRNA response to CSH in culturedastroglia (intense HOS activity present). The glial HO-1 mRNA responseto CSH progressively increases (abrogation of HOS activity) withincreasing dilutions of AD plasma using standard media (lanes 7–15).

Panel A and panel B of FIG. 6 represent data obtained using plasmaobtained from two different AD patients (A: patient 1; B: patient 2). Asnoted above and shown again in FIG. 6, untreated rat astroglia grown instandard culture media exhibit little or no detectable HO-1 mRNA (lane1), however, HO-1 expression increases significantly upon CSH treatment(lane 2). In the absence of CSH treatment, rat astroglia incubated withAD plasma show no detectable HO-1 mRNA (lane 3), also as noted inExample 1. CSH treatment of rat astroglia incubated with undiluted ADplasma (“100%”) failed to induce any significant induction of HO-1expression (lanes 4–6), due to the intense HOS activity present in theundiluted AD plasma. However, the rat astroglial response to CSHprogressively increases (abrogation of HOS activity) with increasingdilutions of the AD plasma using standard media (lanes 7–15). Therefore,there appears to exist in AD plasma a HOS factor whose plasmaconcentration correlates with HOS activity.

EXAMPLE 7 Effect of Heat Treatment on HOS Activity

Plasma HOS activity was assayed via the determination of HO-1 mRNAlevels in treated rat astroglia as described in Example 1. In this case,the effects of prior heat treatment of AD plasma were examined, asdocumented in FIG. 7.

As noted above, control rat astroglia grown in standard media or exposedto human plasma (from normal [NEC] subjects or AD patients) exhibitlittle or no HO-1 mRNA via Northern analysis in the absence of CSHtreatment (lanes 1, 5, 7 and 10). CSH treatment (880 μM CSH×6 h) ofastroglia grown in standard media results in the observation of anintense HO-1 mRNA signal (lane 2). Also as noted above, this inductionof HO-1 expression in response to CSH treatment is significantlyattenuated in astroglia incubated in AD plasma for 24 h (lanes 3 and 4).However, this attenuation is no longer observed when the AD plasma isheated (100 C for 10 min.) prior to incubation with rat astroglia,indicating that as a result of this pre-heating AD plasma HOS activityis abrogated, as observed in the robust HO-1 mRNA signal seen in lane 6.CSH treatment of rat astroglia with normal plasma either untreated orpre-heated results in a robust observed HO-1 mRNA signal, since HOSactivity is absent in either case (lanes 8 and 9). Therefore, these dataindicate that HOS activity in AD plasma is mediated by a protein.

EXAMPLE 8 Partial Purification of HOS Factor by Heparin-Agarose AffinityColumn Chromatography

Plasma from one normal subject (NEC) and one AD patient (AD) wassubjected to affinity purification on a heparin-agarose column asdescribed in Sasaki et al. (1987), herein incorporated by reference, asfollows:

Plasma preparation for loading onto Heparin Agarose column: The NEC andAD plasma tubes were thawed at 4 C. The samples were then dialyzedagainst Heparin Agarose column loading buffer [HALB: 20 mM Hepes (SIGMAChemical Co., Saint Louis, Mich., USA, Catl # H-4034) pH 7.2, 150 mMNaCl, protease inhibitor tablet (Roche Diagnostics, Laval, PQ, CANADACatl. # 1 873 580)] for 2 h with gentle stirring. The samples were thencentrifuged at 15,000 g at 4 C for 20 minutes and supernatantscollected.

Heparin Agarose affinity column chromatography: The Heparin Agarosecolumn (1 cm×2 cm SIGMA Chemical Co., Saint Louis, Mich., USA, Catl #H-0402) was prewashed with 20 ml of HALB. Plasma supernatants wereloaded on the column. The column was washed with 4–6 volumes of HALB and1 ml fractions collected. The flow-through fractions containing proteinwere pooled. The column was eluted with elution buffer [EB: 20 mM HepespH 7.2, 1 M NaCl, protease inhibitor] and 1 ml eluates containingproteins were pooled and dialyzed against HALB for 2–4 h.

The preparation of protein (e.g. plasma or column fractions) for the ratastroglia/HOS activity assay was performed as described in Sasaki et al.(1987), herein incorporated by reference, as follows:

Media was removed from 70 ml, 25 cm² flasks containing confluentastrocyte monolayer (7–10 days in culture). To each individual flask,1.4 ml of NEC or AD plasma was added. To each individual flask,approximately 1.4 ml (10 mg protein based on the Bradford BioRad Proteinassay kit using BSA as control) of the Heparin Agarose flow-throughfraction derived from NEC or AD subjects was added with 0.6 ml ofcomplete DMEM medium. To each individual flask, approximately 1.4 ml(0.5 mg protein) of the Heparin Agarose eluate fractions derived fromNEC or AD subjects was added with 0.6 ml of complete DMEM medium.

Subsequently, the various samples were assayed for HOS activity asdescribed in Example 1. Briefly, the eluate samples were incubated withrat astroglia, which were then subjected to CSH treatment. SubsequentmRNA isolation and Northern analysis to determine the level of HO-1 mRNAwas performed, with the results shown in FIG. 8.

FIG. 8: Panel A: Northern blots of HO-1 mRNA. Panel B: Control GAPDHmRNA. Plasma from one NEC and one AD patient was affinity purified on aheparin-agarose column and the eluate, collected using a high saltsolution, was dialyzed. In the absence of CSH treatment, control ratastroglia pre-incubated with heparin eluates from NEC or AD plasma for24 h did not exhibit an increase in HO-1 expression, as observed by therelatively faint HO-1 mRNA bands which correspond to these samples(lanes 1 and 4). CSH treatment (880 μM CSH×6 h) of rat astrogliaincubated with the heparin eluate from NEC plasma results in aninduction of HO-1 expression, as observed by intense HO-1 mRNA bands(lanes 2 and 3). Conversely, no augmentation of HO-1 mRNA bands inresponse to CSH treatment was observed in rat astroglia incubated for 24h with the heparin eluate fraction derived from the plasma of the ADpatient. These data support the presence of a HOS factor in the plasmaof AD patients, but not normal (NEC) subjects, and indicate that thefactor binds to heparin-agarose affinity columns.

EXAMPLE 9 Further HOS Purification of Heparin Agarose Eluate byConcanavalin-A (Con-A) Agarose Affinity Column Chromatography

Heparin Agarose column eluate (as described in FIG. 8) was dialyzedagainst loading buffer: 50 mM Hepes, pH 7.2 containing 150 mM NaCl, 1 mMMgCl₂, 1 mM MnCl₂, 1 mM CaCl₂ and Complete™ EDTA-free Protease inhibitorcocktail for 4 h at 4° C. The dialysate was loaded onto Con-A Agarosecolumn. The column was washed with four bed volumes of loading buffer.The HOS fraction was eluted with loading buffer containing 0.2Mα-D-methyl mannopyranoside. The eluate was dialyzed against loadingbuffer. The HOS bioassay (glial HO-1 mRNA response to 880 μM CSH×6 h)was performed as described in Example 1 and FIG. 1. Results are shown inFIG. 9. Glial HO-1 mRNA bands were faint in all specimens not exposed toCSH. Robust HO-1 mRNA responses to CSH were observed in controlastroglial cultures (grown in standard culture media) and astrocytesincubated for 24 hours in NEC plasma. In contrast, HO-1 mRNA responsesto CSH were markedly suppressed in astrocytes incubated for 24 hours in(i) whole AD plasma, (ii) dialyzed AD plasma prior to heparin-ConAchromatography and (iii) heparin Agarose-ConA eluate derived from ADplasma These data indicate that the AD plasma HOS factor binds to ConAcolumns and is therefore likely a glycoprotein.

EXAMPLE 10 Further HOS Purification of Heparin Agarosecon-ConavalinEluate Derived from 4 Pooled AD Plasma Samples (29 cc Starting Material)on Superose™ 12 HR FPLC Column

Results are shown in FIGS. 10 to 12. Heparin Agarose—ConA Agarosepurified AD plasma (1-ml) was dialyzed against 20 mM Hepes, pH 7.2containing 150 mM NaCl and Complete™ EDTA-free Protease inhibitorcocktail (1 tablet/100-ml; Catl. # 1873580, Lot 61320101, RocheDiagnostics, Quebec, Canada) for four hours at 4 C. The dialyzedfraction was loaded on Superose™ 12 HR FPLC 1-cm diameter column (Catl.# 17-0538-01, Lot # 8283034) [Amersham Pharmacia Biotech, Inc QuebecCanada]. HOS activity was measured by bioassay in each fraction asdescribed in Example 1 and FIG. 1. As shown in FIG. 10, robust HOSactivity was observed in fraction number 20–22.

FIG. 11: Relative protein concentrations in Superose™ 12 HR FPLC Columnfractions derived from pooled AD plasma samples described in FIG. 10.Each 0.5-ml fraction of the flow-through was collected and absorbance(O.D.) measured at 280 nm by spectrophotometer. A graph of O.D. versusfraction number is plotted. Arrow denotes protein concentration infraction (number 20–22) exhibiting robust HOS activity (FIG. 11).

FIG. 12: A chromatogram from a function test of Superose™ 12 HR FPLC1-cm diameter column (Catl. # 17-0538-01, Lot # 8283034) [AmershamPharmacia Biotech, Inc Quebec Canada] using standard protein mixtures.Plasma samples were derived from the same pooled AD plasmas described inFIG. 10. Each 0.5-ml fraction of the flow-through was collected andabsorbance measured at 280 nm. A graph of peak molecular weight for eachprotein standard versus fraction number was plotted. Based on theelution profile of standards with known molecular weight, the molecularweight of the HOS-positive fraction (number 20–22) is estimated atapproximately 90 kDa.

EXAMPLE 11 Effects of NEC and AD Plasma on Astrocyte HO-1 mRNA Inductionby Multiple Stimuli

The HOS bioassay was performed as described in Example 1 and FIG. 1.Northern blots for HO-1 mRNA (top) and respective GAPDH mRNA (bottom)are shown in FIG. 13. AD plasma strongly suppressed the HO-1 mRNAresponse to CSH (880 μM), interleukin-1β (Il-1β 50 and 100 ng/ml), andhomocysteine (HC 200 μM). NEC plasma showed no HOS activity in the faceof these stimuli. AD plasma completely suppressed, whereas NEC plasmapartially suppressed, the HO-1 mRNA response to tumour necrosis factor-α(TNF-α 50 and 100 ng/ml). AD and NEC plasma exhibited partial and equalsuppression of the glial HO-1 mRNA response to menadione (Mena 100 μM).These data indicate that: A) The HOS protein in AD plasma is activeagainst multiple inducers of astroglial HO-1 mRNA. B) HOS activity in ADplasma is particularly potent in the face of TNF-α challenge. Sincepartial HOS activity against TNF-α also occurs in NEC plasma,differences in HOS protein expression between AD and NEC may bequantitative rather than qualitative. C) Simple antioxidant behaviordoes not account for HOS activity in AD plasma because both AD and NECplasma exhibit partial and equal suppression of the glial HO-1 mRNAresponse to the pro-oxidant, menadione.

The above examples illustrate application of the testing method todetect HOS activity. Further, the above examples illustrate the testingmethod using plasma as the tissue or body fluid sample. The method canalso be applied to other tissue or body fluids such as blood,cerebrospinal fluid, urine, saliva, epithelia and to fibroblast celllines derived from patients.

The test can be applied to compare the level of HOS activity in aspecific patient over a period of time, or to compare the level of HOSactivity in a patient with the corresponding level in a normal controlpopulation.

The above examples further demonstrate the presence of a HOS factorwhich is a glycoprotein, in an embodiment a mannoprotein, having anapproximate molecular weight in the range of 80–100 kDa, in anembodiment having a molecular weight of approximately 90 kDa, and whichis not a glucocorticoid.

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The invention claimed is:
 1. A heme oxygenase-1 suppressor (HOS) factor,said HOS factor having the activity of attenuating an increase in hemeoxygenase-1 (HO-1) occurring in response to exposure to an experimentalagent or treatment of oxidative stress, said HOS factor by: subjecting atissue or body fluid sample derived from a patient suffering from adementing disease to affinity chromatography using one or a sequentialcombination of a heparin-agarose column, a concanavallin-A (Con-A)agarose column, or a Superose™-12 column; eluting fractions of thesample and selecting those fractions containing protein having amolecular weight in the range of 80–100 kDa as determined from anelution profile of protein standards with known molecular weight; andassaying the selected fractions eluted from affinity chromatography forHOS activity by assessing ex vivo the capacity of said fractions toattenuate an increase in the level of heme oxygenase-1 (HO-1) occurringin response to an experimental agent or treatment of oxidative stress,wherein said HOS factor maintains attenuation of HO-1 activity even whenexposed to an antioxidant.
 2. The HOS factor according to claim 1,wherein the eluted fractions of the sample are selected to containprotein having a molecular weight of 90 kDa as determined from anelution profile of protein standards with known molecular weight.
 3. TheHOS factor according to claim 1, wherein the sample is derived from apatient with Alzheimer disease.
 4. The HOS factor of claim 1 wherein theHOS factor substantially binds to the heparin-agarose column underconditions of 20 mM Hepes pH 7.2, 150 mM NaCl, and is substantiallyeluted from the heparin-agarose column under conditions of 20 mM HepespH 7.2, 1M NaCl.
 5. The HOS factor of claim 1, wherein the exposure toan experimental agent or treatment of oxidative stress comprisesexposure to one or more of metal ions, amino acid analogues, sulfhydrylagents, interleukin-1β(IL-1β), tumour necrosis factor-α (TNF-α) orhyperthermia.
 6. The HOS factor of claim 1, wherein the sulfhydryl agentis cysteamine or homocysteine.
 7. The HOS factor of claim 6, wherein thesulfhydryl agent is cysteamine.
 8. A method for assessing dementingdiseases in a patient comprising: determining the level of hemeoxygenase-1 suppressor (HOS) factor or activity, in tissue or a bodyfluid obtained from a patient; and comparing said level of HOS factor oractivity with the corresponding level of HOS factor or activity incorresponding tissue or body fluid obtained from at least one controlperson, whereby if said level of HOS factor or activity is greater thansaid corresponding level of HOS factor or activity in said tissue orbody fluid obtained from at least one control person then said patientsuffers from a dementing disease; wherein such method is used to predictthe onset of, diagnose, or prognosticate dementing diseases.
 9. Themethod according to claim 8 wherein the tissue or body fluid is selectedfrom blood, plasma, lymphocytes, cerebrospinal fluid, urine, saliva,epithelia and fibroblasts.
 10. The method according to claim 8 whereinthe dementing disease is selected from the group consisting of AlzheimerDisease, Age-Associated Cognitive Decline, Mild Cognitive Impairment,Parkinson disease with dementia, Progressive Supranuclear Palsy,Vascular (i.e. multi-infarct) Dementia, Lewy Body Dementia, Huntington'sDisease, Down's syndrome, normal pressure hydrocephalus, corticobasalganglionic degeneration, multisystem atrophy, head trauma,neurosyphilis, Creutzfeld-Jacob disease and other prion diseases, HIVand other encephalitides, and metabolic disorders such as hypothyroidismand vitamin B12 deficiency.
 11. The method of claim 8 wherein thecontrol person is a normal age-matched person.
 12. The method of claim 8wherein the method is used to prognosticate dementing diseases andwherein the control person is the patient from whom the correspondingtissue or body fluid was obtained at another time.
 13. A commercialpackage comprising means for determining the level of HOS factor oractivity as defined in claim 1, in tissue or body fluid obtained from apatient, and instructions for comparing said level of HOS factor oractivity with an established standard of the corresponding HOS activityin a corresponding control tissue or body fluid.
 14. The commercialpackage of claim 13 for use in a method for assessing dementing diseasesin a patient.
 15. A medicament comprising the HOS factor as defined inclaim 1 in combination with a pharmaceutically acceptable carrier, forthe treatment of a dementing disease.